No abstract

We have earlier shown that microglia, the immune cells of the CNS, release microparticles from cell plasma membrane after ATP stimulation. These vesicles contain and release IL-1b, a crucial cytokine in CNS inflammatory events. In this study, we show that microparticles are also released by astrocytes and we get insights into the mechanism of their shedding. We show that, on activation of the ATP receptor P2X 7 , microparticle shedding is associated with rapid activation of acid sphingomyelinase, which moves to plasma membrane outer leaflet. ATPinduced shedding and IL-1b release are markedly reduced by the inhibition of acid sphingomyelinase, and completely blocked in glial cultures from acid sphingomyelinase knockout mice. We also show that p38 MAPK cascade is relevant for the whole process, as specific kinase inhibitors strongly reduce acid sphingomyelinase activation, microparticle shedding and IL-1b release. Our results represent the first demonstration that activation of acid sphingomyelinase is necessary and sufficient for microparticle release from glial cells and define key molecular effectors of microparticle formation and IL-1b release, thus, opening new strategies for the treatment of neuroinflammatory diseases.

Microvesicles (MVs) released into the brain microenvironment are emerging as a novel way of cell-to-cell communication. We have recently shown that microglia, the immune cells of the brain, shed MVs upon activation but their possible role in microglia-to-neuron communication has never been explored. To investigate whether MVs affect neurotransmission, we analysed spontaneous release of glutamate in neurons exposed to MVs and found a dose-dependent increase in miniature excitatory postsynaptic current (mEPSC) frequency without changes in mEPSC amplitude. Paired-pulse recording analysis of evoked neurotransmission showed that MVs mainly act at the presynaptic site, by increasing release probability. In line with the enhancement of excitatory transmission in vitro, injection of MVs into the rat visual cortex caused an acute increase in the amplitude of field potentials evoked by visual stimuli. Stimulation of synaptic activity occurred via enhanced sphingolipid metabolism. Indeed, MVs promoted ceramide and sphingosine production in neurons, while the increase of excitatory transmission induced by MVs was prevented by pharmacological or genetic inhibition of sphingosine synthesis. These data identify microglia-derived MVs as a new mechanism by which microglia influence synaptic activity and highlight the involvement of neuronal sphingosine in this microgliato-neuron signalling pathway.

The authors report that the sequence of an RNA interference oligonucleotide for Mcm21R/CENP-O was incorrectly annotated in the Supplementary Materials and Methods to the article cited above. We had reported the oligonucleotide (ATATGAGTCTGGTCTCCTA) to comprise positions 959-977 of the coding sequence of Mcm21R (as measured from the start codon) whereas it actually lies between positions 884-902. This error arose because several sequences for Mcm21R cDNA versions existed in the NCBI database in 2006. Now that the sequence has been curated, we noticed our error. We apologize for any inconvenience caused.

Duchenne muscular dystrophy is a relatively common disease that affects skeletal muscle, leading to progressive paralysis and death. There is currently no resolutive therapy. We have developed a treatment in which we combined the effects of nitric oxide with nonsteroidal antiinflammatory activity by using HCT 1026, a nitric oxide-releasing derivative of flurbiprofen. Here, we report the results of long-term (1-year) oral treatment with HCT 1026 of two murine models for limb girdle and Duchenne muscular dystrophies (␣-sarcoglycan-null and mdx mice). In both models, HCT 1026 significantly ameliorated the morphological, biochemical, and functional phenotype in the absence of secondary effects, efficiently slowing down disease progression. HCT 1026 acted by reducing inflammation, preventing muscle damage, and preserving the number and function of satellite cells. HCT 1026 was significantly more effective than the corticosteroid prednisolone, which was analyzed in parallel. As an additional beneficial effect, HCT 1026 enhanced the therapeutic efficacy of arterially delivered donor stem cells, by increasing 4-fold their ability to migrate and reconstitute muscle fibers. The therapeutic strategy we propose is not selective for a subset of mutations; it provides ground for immediate clinical experimentation with HCT 1026 alone, which is approved for use in humans; and it sets the stage for combined therapies with donor or autologous, genetically corrected stem cells.HCT 1026 ͉ satellite cells ͉ skeletal muscle ͉ ␣-sarcoglycan-null mice ͉ mdx mice